Nitric oxide (NO), a free radical gas that is short-lived in biological materials, has recently been identified as a molecule that plays a fundamental role in biological processes. As a result, research into the physiology and pathology of nitric oxide has grown. This research activity has created a demand for accurate and precise techniques for the detection and quantification of nitric oxide.
The use of spectrophotometry, chemilluminescence, and paramagnetic resonance methods for detecting nitric oxide in biology and medicine are well established. However, these techniques require that a sample of biological fluid, for example, the extracellular fluid in a tissue or the support buffer in a suspension of cells, must be analyzed removed from its biological context. Measurements made on such samples reflect nitric oxide concentration at a single time, and when assembled in a series make a discontinuous record. Therefore, these methods are not ideal for following rapid processes because changes in nitric oxide concentration are not observed if they occur between sampling points. Moreover, the ability to follow rapid changes in NO concentration is important because nitric oxide is unstable in the presence of oxygen, persisting only a few minutes or seconds in biological systems.
Recently, electrodes for the direct electrochemical detection of nitric oxide have been developed. The earliest of these electrodes, known colloquially as the “Shibuki electrode”, uses a Pt electrode to oxidize nitric oxide at 800 mV and to register the resulting oxidation current. See K. Shibuki Neuroscience Research 1990, 9, 69-76. This sensor is of limited utility because it is subject to the destructive buildup of oxidation products from nitric oxide within the enclosed electrolyte surrounding the Pt electrode. More recently, a method has become available that uses a metalloporphyrin membrane. See T. Malinski et al. Nature, 1992, 358, 676-677; T. Malinski et al, “Nitric Oxide Measurement by Electrochemical Methods”, Methods in Nitric Oxide Research, chapter 22 (1996); and Published International Patent Application No. WO 93/21518. This sensor is constructed by electrochemically depositing a metalloporphyrin, for example nickel-tetrakis (3-methoxy-4-hydroxyphenyl) porphyrin, on a carbon electrode, followed by coating the porphyrin surface with a layer of Nafion™ (Dupont). Direct measurements of nitric oxide with good sensitivity and selectivity have been reported using this porphyrinized electrode. Its disadvantages include the difficulty in handling the fragile micron-diameter carbon fibers, which require manipulation under a microscope with cold illumination (or under water) to eliminate thermal convection currents that disturb the fibers. In another instance, a biochemically-modified electrode has also been proposed that employs Cytochrome C as a nitric oxide sensor. See K. Miki et al. J. Electroanal. Chem., 1993, 6, 703-705. Other methods for nitric oxide detection include using electrodes constructed of gold or platinum (See F. Pariente et al. J. Electroanal. Chem, 1994, 379, 191-197 and F. Bedioui et al. J. Electroanal. Chem, 1994, 377, 295-298) and a porphyrinic-based platinum-iridium electrode. See K. Ichimori et al, “Practical nitric oxide measurement employing a nitric oxide-selective electrode”, Ref. Sci. Instrum., 65 (8) August 1994 and H. Miyoshi, FEBS Letters, 1994, 345, 47-49.
Recent developments in solid state materials technology indicates that these materials may serve a useful role as sensory devices for determining the presence of a variety of analytes. One class of potentially useful solid state materials is conducting polymers. These polymers typically include organic structures possessing a degree of unsaturation to allow electronic communication throughout a polymeric structure. Because polymers in general are synthesized from monomer components, the design of the conducting properties of a conducting polymer can be facilitated by engineering the monomer component to a desired specificity. Moreover, polymers containing both organic and metal ion components afford a larger number of variables over organic-based polymers through the incorporation of a diverse number of metal ions.
A variety of conducting polymers of different compositions and physical properties have been reported. Zotti et al. disclosed in situ conductivity of some polypyrroles and polythiophenes redox modified with pendant ferrocene groups. It was found that the electron hopping rate through the conductive polymer backbone is increased by a decrease of the ferrocene backbone distance and by conjugation of ferrocene with the backbone itself. Zotti et al. Chem. Mater. 1995, 7, 2309. Cameron et al. described a benzimidazole-based conjugated polymer with coordinated [Ru(bpy)2]2+ that provides direct electronic communication between the ruthenium complex and the polymer. Cameron et al. Chem. Commun. 1997, 303. Audebert et al. report a series of conducting polymers based on metal salen containing units comprising mononuclear copperII, cobaltII, nickelII and zincII complexes. Under carefully chosen conditions, thick electroactive polymer deposits are formed upon electrochemical oxidation of the monomer in solution. Audebert et al. New. J. Chem. 1992, 16, 697. U.S. Pat. No. 5,549,851 discusses silicon containing polymers admixed with an amine compound. A highly conductive polymer composition is formed upon doping with an oxidizing dopant, typically iodine and ferric chloride. The composition has improved shapability and can form a highly conductive film or coating.
The integration of receptors into conducting polymer frameworks has been shown to produce materials which provide changes in physical characteristics upon binding of targeted analytes. Devynck et al. described a material containing Co(III) porphyrin sites. Variations in the Co(III)/Co(II) redox couple were observed upon exposure to pyridine and with changing pyridine concentrations. U.S. Pat. No. 4,992,244 discloses a chemical microsensor fabricated using Langmuir-Blodgett techniques. The chemical microsensor was a film based on dithiolene transition metal complexes that displayed differing degrees of current changes upon exposure to varying concentrations of a gas or vapor.
Despite numerous advances in polymeric materials as chemoresistive and sensory devices, there still remains a need to develop sensitive, selective sensors that measure analyte concentration in real time. One promising approach to developing such a sensor is through transition-metal containing conducting polymers. In certain instances, these materials are sensitive to small molecules, such as NO, and hence could be used in detection devices.